Previous work from our group and others have shown that medial temporal lobe (MTL), orbital prefrontal cortex (OFC), and ventral striatum (VS) are interconnected and are important for integration of visual and reward value information. Damage to any of those structures has an adverse effect on the ability to judge relative reward values, that is, after bilateral damage to any of these structures monkeys have difficulty in recognizing which rewards are biggest (best) and which are worth less. In our effort to learn how the signals in these interconnected brain regions interact to give rise to reward expectancy, we are using tools from molecular genetics. We are using nonreplicating viral vectors that carry genes into neurons to introduce functional genes into neurons in localized brain sites. Tonically active interneurons are cholinergic interneurons found throughout the striatum. Despite their sparse distribution (1-2% of striatal neurons) they are presumed to play important functional roles in reward seeking behavior. Our recent recordings of these neurons show that the pattern of their activity is related to the value of a predicted outcome. This response modulation suggests that these neurons are related to the predicted value. To study what these neurons contribute to reward seeking behavior we wish to manipulate their function during behavioral and physiological studies. To do this we are using techniques taken from from modern genetic techniques in old-world monkeys. We have used a modern technique, chemogenetics, to silence neurons in whole brain regions such as the striatum. Given our physiological recording data we would like to have a cell-type specific for tonically active neurons only. Vector mediated RNA interference (RNAi) can in principle be used to target cell-specific pathways. We have made a lentivirus with a human synapsin promoter that co-expresses a microRNA scaffold against the Choline Acetyltransferase (ChAT) mRNA with either mCherry or a hM4Di-CFP fusion protein. To create shRNAs specific to ChAT mRNA, we used the DSIR (Designer of Small Interfering RNA) webtool to rank potential candidates; we then further selected them for targeting all ChAT isoforms and against targeting of other monkey genes. Three candidates were selected, and each was cloned into a microRNA MirE scaffold in the 3 UTRs of the hM4Di-CFP open reading frame (ORF). The best ranked candidate was also cloned into a MirE scaffold of the 3 UTR of an mCherry reporter. In addition, we created a polycistronic array of four different shRNAs in the 3 UTR of either ORF. Two monkeys were injected into multiple sites for each construct. Following a minimum of six weeks, to ensure adequate expression, brain sections were stained with fluorescent antibodies and confocally visualized for ChAT protein and reporter expression. When the ChAT positive cells were counted the neurons in injected regions of the striatum showed significantly less ChAT expression. In the treated region the strength of reporter was inversely proportional to the ChAT expression strength. Thus, we conclude that more reporter indicates stronger anti-ChAT shRNA, and less ChAT. All mirE shRNA constructs showed reporter correlated shifts of varying magnitudes from strong to weak ChAT expressing cells, with one construct also showing a 40% overall reduction in ChAT cells. To our surprise, the polycistronic constructs had no discernible effects. We are awaiting the results of behavioral testing in monkeys treated in the rostro-medial ventral caudate, where silencing of all cells with the DREADD caused a decrease in behavioral reward sensitivity. Because we have coexpressed the shRNA suppressing ChAT activity and a chemogenetic DREADD receptor in monkey striatum, we will be able to compare the effect of exclusively silencing the ChAT neurons with the silencing of all of the neurons. We have recently injected the OFC with a lentivirus virus expressing anterogradely transported green fluorescent protein (GFP) and visualized strong projections to both VS and the MTL, identifying a feedforward connection. To identify the feedback connections from OFC to medial temporal lobe, we injected a retrogradely transported Lentivirus (Lenti-Fug-E-syn::GFP) into the OFC projection site of the feedforward expression. In a second monkey, we injected a retrogradely transported Adeno-Associated Virus (AAV-retro-hSyn-GCaMP). Antibody staining for the GFP reporter expression, followed by immunohistochemistry as well as confocal microscopy, revealed common regions of afferent VS and OFC projections from the basolateral amygdala (BLA) and perirhinal cortex (PR). It also showed that area TE, in the anterior temporal lobe, projects strongly to the OFC while there were few cells projecting to the VS. Conversely, there is a dense entorhinal cortex projection to the VS but not to the OFC. We are now using a two-component retrograde virus system expressing (chemogenetic) receptors that allow chemically activated reversible silencing of targeted neurons to examine the functional properties of the projections reported above. We have now also found that there are single neurons in OFC that project to both to area TE in the base of the superior temporal sulcus and to neurons in the basal lateral nucleus of the amygdala. There are also neurons in the basal lateral nucleus of the amygdala that project to both the OFC and the ventral rostromedial region of the caudate. Thus, it seems that the information presumably related to reward is processed simultaneously in these brain areas.